The present invention relates to the identification of a number of human genes as cellular targets for the design of therapeutic agents for suppressing human immunodeficiency virus (HIV) infection. These genes encode products which appear to be necessary for HIV replication, as evidenced by an inhibition of HIV infection in cells in which the expression of these genes is down-regulated. Therefore, inhibitors of these genes and their encoded products may be used as therapeutic agents for the treatment and/or prevention of HIV infection. In addition, the invention also relates to methods for identifying additional cellular genes as therapeutic targets for suppressing HIV infection, and methods of using such cellular genes and their encoded products in screening assays for selecting additional inhibitors of HIV.
2.1. The Human Immunodeficiency Virus
The primary cause of acquired immunodeficiency syndrome (AIDS) has been shown to be HIV (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503). HIV causes immunodeficiency in an individual by infecting important cell types of the immune system, which results in their depletion. This, in turn, leads to opportunistic infections, neoplastic growth and death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, 1988, Science 240:1427-1439). There are at least two distinct subtypes of HIV: HIV-1 (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503) and HIV-2 (Clavel et al., 1986, Science 233:343-346; Guyader et al., 1987, Nature 326:662-669). Genetic heterogeneity exists within each of these HIV subtypes.
CD4+ T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon viral protein gp120 binding to the cellular CD4 receptor molecule (McDougal et al., 1986, Science 231:382-385; Maddon et al., 1986, Cell 47:333-348).
2.2. HIV Treatment
HIV infection is pandemic and HIV-associated diseases have become a world-wide health problem. Despite considerable efforts in the design of anti-HIV modalities, there is, thus far, no successful prophylactic or therapeutic regimen against AIDS. However, several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al., 1991, FASEB J. 5:2369-2381). For example, virally-encoded reverse transcriptase has been a major focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2, 3xe2x80x2-dideoxynucleotide analogs such as AZT, ddI, ddC, and ddT have been shown to be active against HIV (Mitsuya et al., 1990, Science 249:1533-1544). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al., 1989, Science 243:1731-1734). In addition, these drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.
Another stage of the HIV life cycle that has been targeted is viral entry into the cells, the earliest stage of HIV infection. This approach has primarily utilized recombinant soluble CD4 protein to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). To date, clinical trials of recombinant, soluble CD4 have produced inconclusive results (Schooley et al., 1990, Ann. Int. Med. 112:247-253; Kahn et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
Additionally, the later stages of HIV replication which involve crucial virus-specific secondary processing of certain viral proteins and enzymes have been targeted for anti-HIV drug development. Late stage processing is dependent on the activity of a virally-encoded protease, and drugs including saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al., 1993, Persp. Drug. Discov. Design 1:69-83). With this class of drugs, the emergence of drug resistant HIV mutants is also a problem; resistance to one inhibitor often confers cross resistance to other protease inhibitors (Condra et al., 1995, Nature 374:569-571). These drugs often exhibit toxic side effects such as nausea, altered taste, circumoral parethesias, fat deposits, diarrhea and nephrolithiasis.
Antiviral therapy of HIV using different combinations of nucleoside analogs and protease inhibitors have recently been shown to be more effective than the use of a single drug alone (Torres et al., 1997, Infec. Med. 14:142-160). However, despite the ability to achieve significant decreases in viral burden, there is no evidence to date that combinations of available drugs will afford a curative treatment for AIDS.
Other potential approaches for developing treatment for AIDS include the delivery of exogenous genes into infected cells. One such gene therapy approach involves the use of genetically-engineered viral vectors to introduce toxic gene products to kill HIV-infected cells. Another form of gene therapy is designed to protect virally-infected cells from cytolysis by specifically disrupting viral replication. Stable expression of RNA-based (decoys, antisense and ribozymes) or protein-based (transdominant mutants) HIV-1 antiviral agents can inhibit certain stages of the viral life cycle. A number of anti-HIV suppressors have been reported, such as decoy RNA of TAR or RRE (Sullenger et al., 1990, Cell 63:601-608; Sullenger et al., 1991, J. Virol. 65:6811-6816; Lisziewicz et al., 1993, New Biol. 3:82-89; Lee et al., 1994, J. Virol. 68:8254-8264), ribozymes (Sarver et al., 1990, Science 247:1222-1225; Wecrasinghe et al., 1991, J. Virol. 65:5531-5534; Dropulic et al., 1992, J. Virol. 66:1432-1441; Ojwang et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10802-10806; Yu et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6340-6344; Yu et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 92:699-703; Yamada et al., 1994, Gene Therapy 1:38-45), antisense RNA complementary to the mRNA of gag, tat, rev, env (Sezakiel et al., 1991, J. Virol. 65:468-472; Chatterjee et al., 1992, Science 258:1485-1488; Rhodes et al., 1990, J. Gen. Virol. 71:1965. Rhodes et al., 1991, AIDS 5:145-151; Sezakiel et al., 1992, J. Virol. 66:5576-5581; Joshi et al., 1991, J. Virol. 65:5524-5530) and transdominant mutants including Rev (Bevec et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:9870-9874), Tat (Pearson et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:5079-5083; Modesti et al., 1991, New Biol. 3:759-768), Gag (Trono et al., 1989, Cell 59:113-120), Env (Bushschacher et al., 1995, J. Virol. 69:1344-1348) and protease (Junker et al., 1996, J. Virol. 70:7765-7772).
Antisense polynucleotides have been designed to complex with and sequester the HIV-1 transcripts (Holmes et al., WO 93/11230; Lipps et al., WO 94/10302; Kretschmer et al., EP 594,881; and Chatterjee et al., 1992, Science 258:1485). Furthermore, an enzymatically active RNA, termed ribozyme, has been used to cleave viral transcripts. The use of a ribozyme to generate resistance to HIV-1 in a hematopoietic cell line has been reported (Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA 89:10802-06; Yamada et al., 1994, Gene Therapy 1:38-45; Ho et al., WO 94/26877; and Cech and Sullenger, WO 95/13379). In preclinical studies, RevM10, a transdominant Rev protein, has been transfected ex vivo into CD4+ cells of HIV-infected individuals and shown to confer survival advantage over cells transfected with vector only (Woffendin et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2889-2894).
2.3. Cellular Genes Necessary for HIV Replication
Evolution of an intracellular pathogen has resulted in the development of interactions of its genes and gene products with multiple cellular components. For instance, the interactions of a virus with a host cell involves binding of the virus to a specific cellular receptor(s), translocation through the cellular membrane, uncoating, replication of the viral genome, transcription of the viral genes, etc. Each of these events occurs in a cell and involves interactions with a cellular component. Thus, the life cycle of a virus can be completed only if the cell is permissive. Availability of amino acids and nucleotides for replication of the viral genome and protein synthesis, energy status of the cell, the presence of cellular transcription factors and enzymes all contribute to the propagation of the virus in the cell. Consequently, the cellular components, in part, determine host cell susceptibility to infection, and may be used as potential targets for the development of new therapeutic interventions. In the case of HIV, one cellular component which has been used towards this end is the cell surface molecule for HIV attachment, CD4.
Recently, it was reported that HIV entry into a susceptible cell requires the expression of a second receptor, the chemokine receptors (CCR2, CCR3, CCR5 or CXCR4), in addition to CD4 (Moore, 1997, Science 276:51-52). A chemokine receptor normally binds RANTES, MIP-1xcex1 and MIP-1xcex2 as its natural ligand. In the case of HIV infection, it has been proposed that CD4 first binds to gp120 of HIV on the cell surface followed by the binding of the complex to a chemokine receptor to result in viral entry into the cells (Cohen, 997, Science 275:1261). Therefore, chemokine receptors may present an additional cellular target for the design of HIV therapeutic agents. Inhibitors of HIV/chemokine receptor interactions are being tested as anti-HIV agents. However, there remains a need for the discovery of additional cellular targets for the design of anti-HIV therapeutics, particularly intracellular targets for disrupting viral replication after viral entry into a cell.
The present invention relates to compositions and methods for inhibiting HIV infection by down-regulating the expression and/or function of certain human cellular genes. In particular, it relates to a number of human cell-derived polynucleotides which inhibit HIV replication in susceptible cells. The isolated polynucleotides correspond to a portion of a cellular gene or a complement thereof, and are referred to herein as genetic suppressor elements (GSEs). The cellular genes encode intracellular products which are necessary for a productive HIV infection. Additionally, small molecule inhibitors of the same cellular genes and their encoded products are also within the scope of the present invention. The invention also relates to methods for identifying additional cellular genes as therapeutic targets for suppressing HIV infection, and methods for using such cellular genes and their encoded products for selecting additional inhibitors of HIV.
The invention is based, in part, on the Applicants"" discovery that polynucleotides isolated from human cells can prevent the activation of latent HIV-1 in a CD4+ cell line as well as productive HIV infection, and that such polynucleotides correspond to fragments of certain human cellular genes. In that connection, any cellular or viral marker associated with HIV replication can be used to select for such polynucleotides or GSEs. An example of such a marker is CD4, which is conveniently monitored by using a specific antibody.
Based on substantial sequence identity (90%-100%), a number of the isolated GSEs correspond to portions of human cellular genes which encode different subunits of a mitochondrial enzyme complex, NADH dehydrogenase. In addition, inhibitors of this enzyme also inhibit HIV replication in susceptible host cells, including freshly isolated human CD4+ T cells. Furthermore, additional GSEs have been selected which have substantial sequence identity (90%-100%) with human cellular genes which encode 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidylinositol 3 kinase and elongation factor 1 alpha.
Among the GSEs selected to inhibit HIV replication, several function in the sense orientation, while others function in the antisense orientation. Not intending to be bound by any particular theory, the GSEs of the invention are believed to down-regulate a cellular gene by different mechanisms. The GSEs are expressed in a host cell by encoding RNA molecules that may or may not encode polypeptide products. GSEs in the sense orientation may exert their effects as transdominant mutants or RNA decoys. Transdominant mutants are expressed polypeptides which competitively inhibit the normal function of a wild-type protein in a dominant fashion. RNA decoys are protein binding sites that titrate out these proteins. GSEs in the antisense orientation may exert their effects as antisense RNA; i.e. polynucleotides complementary to the mRNA of the target gene. These polynucleotides bind to mRNA and block the translation of the mRNA. Some antisense polynucleotides may act directly at the DNA level to inhibit transcription. The down-regulation of a cellular gene by a GSE, in turn, removes a cellular component necessary for HIV replication, resulting in an inhibition of HIV infection.
A wide range of uses are encompassed by the invention including, but not limited to, HIV treatment and prevention by transferring GSEs as pharmaceutical compositions into HIV-susceptible cell types. For example, GSEs may be transferred into T cells, particularly CD4+ T cells which are the major cell population targeted by HIV. Alternatively, GSEs may be transferred into hematopoietic stem cells in vitro followed by their engraftment in an autologous or histocompatible or even histoincompatible recipient. In another embodiment, any cells susceptible to HIV infection may be directly transduced or transfected with GSEs in vivo. In yet another embodiment, inhibitors of NADH dehydrogenase, 2-oxoglutarate dehydrogenase, M2-type pyruvate kinase, calnexin, ADP-ribosylation factor 3, eukaryotic initiation factor 3, protein tyrosine phosphatase, herpesvirus-associated ubiquitin-specific protease, eukaryotic initiation factor 4B, CD44, phosphatidylinositol 3-kinase and elongation factor-1 alpha may be used as pharmaceutical compositions in vivo to suppress HIV infection.